JP3098640B2 - Immunoassay for human 72-kDa gelatinase / type IV collagenase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an immunoassay for human 72-kDa gelatinase / type IV collagenase (human pro-MMP-2) used in the medical and physiological fields. More specifically, the present invention provides a monoclonal antibody that specifically binds to a specific amino acid sequence of human 72-kDa gelatinase / type IV collagenase (hereinafter referred to as human pro-MMP-2) or to purified human pro-MMP-2. And a method for immunologically quantifying human pro-MMP-2 using the same.

[0002]

BACKGROUND ART The extracellular matrix is composed of an adhesive glycoprotein with collagen, proteoglycan, elastin, fibronectin and laminin (Shimooka et al., Clinical Laboratory, 34, 1719-1724). Matrix metalloproteases (MMPs) play an important role in the degradation of these matrix components. Among them, 72-kDa (kilodalton) gelatinase / type IV collagenase, referred to as pro-MMP-2, is used in human rheumatoid synovial cells (Okada et al., Eur. J. Bioc).
hem. , 194: 721-730, 1990) and Nakajima et al. (Experimental Medicine, 7, 32-40 (542-550), 1).
989), human A205
8 melanoma cells, rat breast cancer cells, human colon cancer cells,
Produced by human bronchial epithelial cells transformed with H-ras and the like, and activated by enzymatic limited degradation or thiol-reactive organomercury compounds such as mercury 4-aminophenylacetate (APMA), gelatin, IV
It has been shown to have the activity of degrading type collagen and type V collagen, as well as proteoglycan core protein, fibronectin, and insoluble elastin (Okada et al., Eur. J. Biochem., 19).
4: 721-730, 1990).

[0003] As described above, human pro-MMP-2 is known as an enzyme (type IV collagenase) that degrades gelatin and type IV collagen, and is described by Wacher et al.
Immunol. Meth. , 126: 239-24.
5, 1990), using a rabbit polyclonal antibody against a synthetic peptide at the N-terminus of human MMP-2, quantifies human MMP-2 by a substrate capture immunoassay. In this method, a method is employed in which a substrate (gelatin) of type IV collagenase is adsorbed on a solid phase, reacted with an antigen in a sample, and further reacted with the antigen using two types of polyclonal antibodies. Zucker et al. (J. Immunol. Met)
h. , 148: 189-198, 1992) are human M
Rabbit polyclonal antibody against MP-2 and human M
Using a mouse monoclonal antibody against MP-2,
The amount of human MMP-2 is quantified by the sandwich type EIA method. In this method, a rabbit polyclonal antibody is used as a solid phase antibody, a sample is added to the antibody, reacted with the antigen in the sample, a mouse monoclonal antibody is added, and then a biotinylated goat anti-mouse immunoglobulin and alkaline phosphatase label The reaction is completed using streptavidin. However, since these methods reported so far use polyclonal antibodies, they are extremely inferior in accuracy and have a long reaction time (eg, 7
Time or 5 hours), and both use two kinds of polyclonal antibodies, so that the quantitative operation becomes complicated and the sensitivity obtained is low.

[0004] The present inventors have previously succeeded in providing a monoclonal antibody against human MMP-2 polypeptide (Japanese Patent Laid-Open No. Hei 4-183397). However, the resulting monoclonal antibody has a low affinity for MMP-2 because the immunogen is a peptide,
Many showed cross-reactivity with MMPs other than P-2, and the quantification method of the sandwich assay system using these monoclonal antibodies had low measurement sensitivity.

An object of the present invention is to use two types of monoclonal antibodies that specifically bind to human pro-MMP-2 to determine human pro-MMP-2 in a test sample with excellent sensitivity and accuracy, and to quickly quantify it. It is to provide a method that can do this. It is still another object of the present invention to provide a diagnostic agent capable of diagnosing diseases such as arthropathy, hyperthyroidism, various cancers and metastatic cancers using the above-mentioned quantitative method.

[0006]

DISCLOSURE OF THE INVENTION The present invention relates to two types of monoclonal antibodies that specifically bind to human 72-kDa gelatinase / type IV collagenase, as an antibody to be bound to a solid support or an antibody to be labeled. Human pro-MMP-2, characterized in that human pro-MMP-2 is immunologically measured by a sandwich method using an antibody recognizing the N-terminal region of human 72-kDa gelatinase / type IV collagenase for any one of them. The present invention provides a method for quantification.

The method of the present invention uses a monoclonal antibody that specifically binds to a different antigenic determinant of human MMP-2, as an antibody to be bound to a solid support or an antibody to be labeled. It is characterized by the following.

In the quantification method of the present invention, an immunological measurement method is used. In this case, the solid support is a polystyrene, polycarbonate, polypropylene, or polyvinyl ball that can adsorb proteins such as antibodies well. Various materials and forms such as microplates, sticks, fine particles or test tubes can be arbitrarily selected and used. On the other hand, as an antibody to be labeled, DEAE-Sep
An anion exchange gel and an IgG fraction, such as hacel, and a specific binding portion Fab ′ obtained by digestion and reduction after pepsin digestion can be used. Examples of the labeled substance in these cases include enzymes (peroxidase, alkaline phosphatase, β-D-galactosidase, etc.), chemical substances, fluorescent substances, radioisotopes, and the like. Hereinafter, the present invention will be described specifically with reference to examples.

Example 1 Preparation of Antigen 1) Preparation of Human MMP-2 Polypeptide Biol. Che
m. 263, 6579-6587, 1988. The amino acid sequence of Wilhelm et al. Each of the human MMP-2 polypeptides (P-1 to P-5) shown in Table 1 was synthesized using a peptide synthesizer 9600 (Millidiene / Biosearch). In addition, cysteine was introduced at the C-terminal of each peptide. Synthetic peptides having a purity of about 70% or less were obtained using μ Bondasphere (5 μ, C18-
(100 °, Waters) column.

2) Preparation of immunogen 2 mg bovine serum albumin (BSA) was added to 1 ml of 0.1 M
1.85 dissolved in phosphate buffer (pH 7.0)
mgN- (ε-maleimidocaproyloxy) succinimide dissolved in 200 μl of dimethylformamide was mixed and incubated at 30 ° C. for 30 minutes. Next, the above mixture was added to a 0.1 M phosphate buffer (pH
7.0) and applied to a PD-10 column (Sephadex G-25M, Pharmacia) equilibrated in B) to which maleimide-bound BSA was collected and concentrated to 1.5 ml or less.
The 50-fold molar amount of each human MMP-2 polypeptide synthesized in the above (a) with respect to the maleimide-bound BSA was
It was mixed with a solution dissolved in 0.1 ml of a 0.1 M phosphate buffer (pH 7.0). After incubation at 4 ° C for 20 hours, an MMP-2 polypeptide-BSA complex was prepared.

3) Human skin fibroblast-derived pro-MMP
Preparation of -2 (a) Cell culture MEM Eagle medium: Minimum Essen
Tial Medium Eagle (modify
d) with Earle's salts (Flow
Lab. ), Add sodium bicarbonate (24 mM)
PH was adjusted to 7.2 with 1N sodium hydroxide or 1N hydrochloric acid.
And sterilized and filtered through a 0.2 μm Toyo membrane filter. At the time of use, additional non-essential amino acids are added
The EM Eagle medium was used.

Human dermal fibroblasts CCD-41SK (American Type Culture Collection) were cultured in MEM Eagle's medium containing 10% fetal calf serum (FCS).
The cells were cultured at 7 ° C. in the presence of 5% carbon dioxide for 3 to 4 days. The culture supernatant was discarded, and an appropriate amount of 10 mM phosphate buffer (pH.4) (PBS) containing 0.1 M sodium chloride was added, followed by gentle shaking and washing. Next, 0.125% trypsin and 0.01% sodium ethylenediaminetetraacetate (EDT
PBS containing A) was added, and the cells were detached from the culture flask by gently shaking, and then MEM Eagl containing 10% FCS was added.
An appropriate amount of e-medium was added. After centrifugation, discard the supernatant and remove 10% FC
An appropriate amount of S-containing MEM Eagle medium was added to suspend the cells. Next, add about 2 × 10 ⁵ cells / m to a new culture flask.
l of cells were added to the mixture at 37 ° C in the presence of 5% carbon dioxide.
Cultured for 4 days.

(B) Cell stimulation Interleukin 1α (I) was added to the cultured cells obtained in (a).
L-1α) acts to stimulate the cells. Decant the culture solution cultured in (a) above, and then
ml of MEM Eagle's medium, add lactalbumin hydrolyzate (GIBCO) and IL-1α (Genzy).
me) was added to a final concentration of 0.2% and 10 U / ml, respectively. 7-1 at 37 ° C in the presence of 5% carbon dioxide
After allowing to stand for 0 days, the culture supernatant was collected and used as a material for preparing human pro-MMP-2.

(C) Purification of human pro-MMP-2 Ammonium sulfate was added to the culture supernatant prepared in the previous section (b) so that the final concentration was 80% saturated, and the mixture was stirred and centrifuged. 50 mM Tris-HCl buffer (pH 7.0, Tris-Ca Buf) containing sodium chloride, 10 mM calcium chloride, and 0.05% polyoxyethylene lauryl alcohol ether (Bridge-35)
fer) was added and dissolved, and Tris-Ca Buffer was added.
dialyzed against r. The solution after dialysis was used for Gelatin-
agarose (Sigma), and the adsorbed protein was subjected to Tris containing 5% dimethylsulfoxide (DMSO).
It was eluted with s-Ca Buffer. Elution fraction is Tri
It was dialyzed against s-Ca Buffer. Next, in order to remove the contaminating TIMP-2, an anti-human TIMP-2 monoclonal antibody (clone No. 68-6H4, deposit number of FERMP-12691) bound Sephapar.
The sample was applied to an oose 4B column, and a fraction passing through the column was collected. The anti-TIMP-2 monoclonal antibody used here was a human TIMP-2 polypeptide (DSGNDIYG
NPIKRIQ) and keyhole limpet hemocyanin (K) as a carrier protein of the immunogen
LH) was prepared according to the method for preparing an anti-human MMP-2 polypeptide monoclonal antibody described below, except that LH) was used. Among the obtained monoclonal antibodies, clone N
o. The antibody from 68-6H4 was conjugated to antibody-conjugated Sepharo.
Used for se 4B column.

Further, in order to remove fibronectin,
The mixture was applied to a Sepharose 4B column conjugated with an anti-fibronectin antibody (Cappel Lab.), And the flow-through fraction was collected. This fraction contained pro-MMP-2, and it was confirmed that it was purified almost solely by polyacrylamide gel electrophoresis (SDS-PAGE) containing sodium dodecyl sulfate. On the other hand, anti-human TIMP
-2 antibody (clone No. 68-6H4) conjugated Seph
The fraction eluted with an arose 4B column using a 0.2 M glycine-HCl buffer (pH 2.5) containing 0.15 M sodium chloride and 10 mM calcium chloride was subjected to SDS-
It is considered that pro-MMP-2 and TIMP-2 were present on PAGE, and that a complex of pro-MMP-2 and TIMP-2 was included. For human pro-MMP-2 for preparing a monoclonal antibody, an eluted fraction of gelatin-agarose was used, and in a one-step sandwich enzyme immunoassay (EIA) described in Example 6 (b) below, anti-human TIMP- 2 Sephalar binding monoclonal antibody
Pro M of the flow-through fraction of the ose 4B column and the Sepharose 4B column conjugated with anti-fibronectin antibody
MP-2 was used as a standard antigen.

Example 2 Preparation of Anti-Human MMP-2 Polypeptide Monoclonal Antibody (a) Immunization Method and Preparation of Spleen Cells 250 μg of the polypeptide-BSA complex prepared by the method described in Examples 1 and 2) was used. Eight weeks-old Balb / c female mice were intraperitoneally administered together with complete Freund's adjuvant for the first immunization. 15 days later, polypeptide-BSA dissolved in 0.1 M phosphate buffer (pH 6.0)
200 μg of the complex was intraperitoneally administered to mice that had been initially immunized, and boosted. Further 38 days later, 70 μg of the polypeptide-BSA complex was intravenously and intravenously administered as in the booster.
30 μg was intraperitoneally administered for final immunization. Three days later, the spleen was removed and a spleen vesicle suspension was prepared.

(B) Cell fusion (1) Material RPMI 1640 medium: RPMI 1640 (Flo
w Lab. ), Sodium bicarbonate (24 mM), sodium pyruvate (1 mM), potassium penicillin G (50 U / ml), streptomycin sulfate (50 μg)
/ Ml) and acacin sulfate (10 µg / ml), the pH was adjusted to 7.2 with dry ice, and the mixture was sterilized and filtered through a 0.2 µm Toyo membrane filter. NS-1 medium: FCS that had been sterilized and filtered was added to the above RPMI 1640 medium to a concentration of 15% v / v).

PEG 4,000 solution: RPMI 16
In a 40 medium, polyethylene glycol 4,000 (PEG
4,000, Merck & Co. ) To 50% (w /
w) to prepare a serum-free solution.

8-azaguanine-resistant myeloma cell SP
2 (SP2 / 0-Ag14) was fused with Select
ed Method in Cellular Imm
unology (eds.BB Mischel a
nd S.D. M. Shiigi), W.M. H. Freem
and and Company (1980), 351-
372 described above.

(2) Cell fusion method The nucleated spleen cells prepared in the above (a) (viable cell rate: 100
%) And myeloma cells (100% viable cell rate)
At a rate of The spleen cells and myeloma cells were separately washed with the above-described RPMI 1640 medium, then suspended in the same medium and mixed at the above ratio for fusion. Using a polypropylene centrifuge tube (Iwaki Glass) having a capacity of 250 ml, 400 × g in 40 ml of RPMI 1640 medium,
After centrifugation for 10 minutes, the supernatant was completely sucked out. To the precipitated cells, 6.0 ml of a PEG 4,000 solution heated at 37 ° C. was dropped dropwise over 1 minute while gently stirring, and further stirred for 1 minute to resuspend and disperse the cells. Next, RPMI heated at 37 ° C
6.0 ml of 1640 medium was added dropwise over 1 minute. After repeating this operation once more, 42.0 ml of the same medium was added to 2 to 2 ml.
The cells were added dropwise over 3 minutes with constant stirring to disperse the cells. This was centrifuged at 400 × g for 10 minutes, and the supernatant was completely removed by suction. Next, 60 ml of 37 ° C. warmed NS-1 medium was quickly added to the precipitated cells, and a large lump of cells was added to 10 ml.
Disperse by careful pipetting using a pipette. Further, 120 ml of the same medium was added for dilution, and 6.0 × 10 ⁵ cells / 0.1 ml of NC cells were added per well to a polystyrene 96-well microwell (Iwaki Glass). The above microwell containing cells was added to 7% carbon dioxide / 93%
The cells were cultured in air at 37 ° C. and 100% humidity.

(C) Selective Propagation of Hybridomas in Selection Medium (1) Medium Used HAT medium: In addition to the NS-1 medium described in (b) above, hypoxanthine (100 μM) and aminopterin (0.
4 μM) and thymidine (16 μM) were added. HT medium: HAT as described above except that aminopterin was removed.
It has the same composition as the medium.

(2) Selection of Hybridoma The next day (day 1) after the start of the culture in (b), two drops (about 0.1 ml) of HAT medium were added to the cells using a Pasteur pipette. On days 2, 3, 5, 8, and 11, half of the medium (0.1 m
I) was replaced with fresh HAT medium and on day 14 half of the medium was replaced with fresh HT medium. Thereafter, every three to four days, half of the medium was replaced with fresh HT medium. Usually, sufficient hybridoma growth is observed in about 2 weeks. The solid phase described in the following item (d) for all hybridoma-grown wells
Positive wells were checked by antibody binding test (ELISA). Then the HT medium 1ml containing 10 ⁷ mouse thymocytes as feeder used after added to a polystyrene 24-well cell well (Iwaki Glass), it was transferred the entire contents of each positive hybridoma has been detected above. This was carried out in the presence of 7% carbon dioxide as in (b) above.
The cells were cultured at 7 ° C. for about one week. During that time, 0.5 ml of the supernatant of each well was replaced with 0.5 ml of fresh HT medium once or twice. When the hybridomas had grown sufficiently, the positivity was confirmed again by ELISA, and each was cloned by the limiting dilution method described in the following section (e). In addition, the residual solution after use for cloning was 25 cm ² of polystyrene.
The sample was transferred to a tissue culture flask (Iwaki Glass) to prepare a sample for cryopreservation.

(D) Anti-human MMP by ELISA
Search for hybridomas producing polypeptide-2 antibody Anal. Biochem. 104, 205-214
(1980) described in Rennerd et al. This method is suitable for detecting a hybridoma antibody. A 96-well microtitration plate (FlowLab.) Was loaded with 100 ng of each human MM.
Coated with P-2 polypeptide, then uncoated portions were blocked with 1% BSA. A portion of the supernatant of the hybridoma growth well obtained in the above (c) was added thereto, and the mixture was incubated at room temperature for about 1 hour. Horseradish peroxidase-labeled goat anti-mouse immunoglobulin (Cappel Lab.) Was added as a secondary antibody, and further added at room temperature for about 1 hour.
Incubated for hours. Next, the degree of brown color generated by adding hydrogen peroxide and o-phenylenediamine as substrates is measured using a microplate reader (MPR-A4, Toyo Soda).
Was used to determine the absorbance at 492 nm to make a determination.

(E) Cloning After the above operation (c), since there is a possibility that two or more types of hybridomas may be growing in each well, cloning is performed by the limiting dilution method to obtain monoclonal antibody-producing hybridomas. get. The cloning medium containing 10 ⁷ mouse thymocytes were prepared as NS-1 medium 1ml per feeder, 36 wells of a 96-well micro-well, five 36-well and 24-well per one and 0.5 hybridomas added. On day 5 and day 12, about 0.1 ml of NS-1 medium was added to all wells. 14 to 15 days after the start of the cloning, sufficient hybridoma growth was observed, and 5
The ELISA method was performed for the group having 0% or more. If all the tested wells are not positive, the number of colonies in the antibody-positive well is confirmed, and 4 to 6 wells in which one colony is confirmed in the wells are selected and recloned. Finally, as shown in Table 2, a hybridoma producing a monoclonal antibody against the human MMP-2 polypeptide was obtained.

(F) In vitro growth and in vivo growth of monoclonal antibody The monoclonal antibody is grown by a conventional method. That is, each of the obtained hybridomas is cultured in an appropriate culture medium such as NS-1 medium (in vitro propagation), and the culture supernatant is used for 10 to 10 minutes.
A monoclonal antibody having a concentration of 100 μg / ml was obtained. On the other hand, in order to obtain a large amount of antibodies, animals of the same strain as those derived from spleen cells and myeloma cells (Balb / c
Mice) were intraperitoneally administered with 0.5 ml of a tumor formation promoter pristane (2,6,10,14-tetramethylpentadecane, Aldrich Chem.) Per mouse. After 1 to 3 weeks, 1 × 10 ⁷ hybridomas were similarly intraperitoneally administered, and 1 to 2 weeks later, ascites containing 4 to 7 mg / ml of the monoclonal antibody produced in vivo was obtained. .

(G) Heavy chain and light chain of monoclonal antibody According to the above-mentioned ELISA method, the culture supernatant of each of the monoclonal clones obtained in (e) above was placed on a microtitration plate coated with human MMP-2 polypeptide. Was added. Next, after washing with PBS, an isotype-specific rabbit anti-mouse Ig antibody (Zymed Lab.)
Was added. After washing with PBS, horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) antibody was added,
Hydrogen peroxide and 2,2'-azino-di (3
-Ethyl benzothiazoline sulfate) to determine the respective heavy and light chains. Table 2 summarizes the results.

(H) Purification of Monoclonal Antibody Each ascites fluid obtained in the above (f) was fractionated with 40% saturated ammonium sulfate, and then purified with IgG1 class antibody.
It is adsorbed to a protein A Affigel (Bio-Rad) column equilibrated with 1.5 M glycine-NaOH buffer (pH 8.9) containing 5 M sodium chloride, washed with the above washing solution, and then 0.1 M citrate buffer (pH 5. Purified by eluting with 0).

Example 3 Preparation of anti-human pro-MMP-2 monoclonal antibody (a) Immunization method and preparation of spleen cells CCD-4 purified by the method described in Examples 1, 3)
It was prepared according to the method of Example 2, (a) using 31 μg of human pro-MMP-2 derived from 1SK cells. In this method, 15 days after the initial immunization, 35 μg of human pro-MMP-2 dissolved in 20 mM phosphate buffer (pH 7.0) containing 0.15 M sodium chloride was intraperitoneally administered, and boosted. In addition, human pro-M
MP-2, 40 μg, was administered intravenously.

(B) Cell fusion The nucleated spleen cells prepared in the above (a) (viable cell rate 100
%) In the same manner as in Examples 2 and (b).

(C) Selective Propagation of Hybridoma in Selection Medium The day after the start of the culture in (b) (day 1), the cells were subjected to the method described in Examples 2 and (c).

(D) Anti-human MMP by ELISA
-2 Search for antibody-producing hybridomas The search was carried out in the same manner as in Example 2, (d).
In this method, a 96-well microtitration plate was coated with 50 ng of human pro-MMP-2.

(E) Cloning The cloning was carried out in the same manner as described in Example 2 and (e).
Finally, monoclonal antibody-producing hybridomas against human pro-MMP-2 derived from CCD-41SK cells shown in Table 3 were obtained.

(F) In vitro growth and in vivo growth of the monoclonal antibody The same procedure as described in Example 2, (f) was performed. (G) Heavy chain and light chain of monoclonal antibody According to the above-described ELISA method, the culture supernatant of each of the monoclonal clones obtained in (e) above was added to a microtitration plate coated with human pro-MMP-2. It carried out similarly to the method described in 2, (g). The results are shown in Table 3. (H) Purification of monoclonal antibody The purification was performed in the same manner as in the method described in Example 2 and (h).

Example 4 Selection of anti-human MMP-2 polypeptide monoclonal antibody and anti-human pro-MMP-2 monoclonal antibody (a) Preparation of materials DMEM medium: Dulbecco's Modifier
d Eagle Medium “Nisui” (Nissui Pharmaceutical) was added with sodium bicarbonate (31 mM) and L-glutamine (5 mM), adjusted to pH 7.2 with dry ice, and sterilized and filtered through a 0.2 μm Toyo membrane.

To select monoclonal antibodies to the human MMP-2 polypeptide, human rheumatoid arthritis (RA) synovial cells were cultured in DMEM medium containing 15% FCS in a 5% CO ₂ incubator at 37 ° C. for 6-7 days. After culturing and centrifuging the cells, 0.2% lactalbumin hydrolyzate and 20 units / ml Tumor Necro
The cells were suspended in a DMEM medium containing sis Factor (TNFα. Genzyme) and cultured similarly for 6 to 8 days. After centrifugation, the supernatant was concentrated by ultrafiltration or 3% trichloroacetic acid (TCA) to obtain a sample for immunoblotting.

The human pro-M derived from CCD-41SK cells prepared by the method described in the above Examples 1 and 3)
MP-2 was used as a sample for immunoblotting to select a monoclonal antibody against human pro-MMP-2.

In order to examine the cross-reactivity with other MMPs or other proteins, human fibrosarcoma cells HT-
1080 (American Type Culture Collection)
In the NS-1 medium in a 5% CO ₂ incubator,
After culturing at 37 ° C. for 2 to 3 days, the cells after centrifugation were lysed with 2% lactalbumin hydrolyzate and 100 units / ml TN
Suspended in RPMI 1640 medium containing Fα,
Cultured for ~ 10 days. The centrifuged supernatant at 700 to 800 rpm for 3 minutes was collected and concentrated by ultrafiltration or 3% TCA to obtain a sample for immunoblotting.

(B) Immunoblotting The samples prepared in Examples 4 and (a) were subjected to SDS-PAGE, and then subjected to cell engineering 1 & 2, 1061-1068 (19).
83), Western blotting was performed according to the method of Tabe described in the above, and after reaction with the culture supernatant of each monoclonal clone, a peroxidase-labeled goat anti-mouse immunoglobulin (Ca
ppel Lab. ) And immunoblotting was performed by an indirect method.

As a result, the anti-human MMP-
Among the two polypeptide monoclonal antibodies, 34-2H
11, 34-27A5, 35-3F2, 39-1H9,
39-4E4, 39-11D11, 39-12B7,3
9-18F3, 42-2H2, 42-5D11, 42-
14H5, 43-3F9, 45-2H8, 45-6F1
2,45-14A8,45-15F9 and 45-17
Seventeen monoclonal antibodies of D8 reacted with pro-MMP-2 derived from human RA synovial cells. Of the anti-human pro-MMP-2 monoclonal antibodies listed in Table 3, 75-
Only 7F7 is human pro-MMP derived from CCD-41SK cells
-2.

The molecular weight of human pro-MMP-2 is TNFα
From the results of immunoblotting using a sample prepared from the human RA synovial cell culture solution stimulated with
-2 was 72 kDa, and active MMP-2 activated by APMA was 67 kDa.

(C) Specificity Each positive anti-human M which was positive by the immunostaining of (b) above
The MP-2 polypeptide monoclonal antibody and the anti-human pro-MMP-2 monoclonal antibody are
Alternatively, to determine if they cross-react with other proteins, human RA synoviocytes stimulated with TNFα and HT1
The specificity of each monoclonal antibody was examined by immunoblotting using a sample prepared from each culture of 080 cells (Table 4).

Among the positive monoclonal antibodies (b), 34-2H11, 39-1H9 and 42-14H
5 showed a cross-reactivity with 92 kDa gelatinase (human pro-MMP-9), indicating that 42-2H
2 and 42-14H5 monoclonal antibodies are human pro-stromal collagenase (human pro-MMP-1) and human prostromulysin-1 (human pro-MMP-
Cross-reaction with 3) was shown. Other monoclonal antibodies were shown to react specifically with human pro-MMP-2 without reacting with other human pro-MMPs or other proteins in cell culture.

Furthermore, among the IgG1 antibodies which were confirmed to react specifically, those having high reactivity with human proMMP-2 were three clones of 43-3F9, 42-5D11 and 75-7F7. It was found to be an antibody.

Example 5 An enzyme-labeled antibody (IgG-POD)
Preparation of complex) 1) Preparation of SH group-labeled IgG Immunoassay 4,209-327,1
983, a mouse anti-human pro-MMP-2 IgG-POD complex was prepared according to the method of Ishikawa et al. A monoclonal antibody (IgG: clone Nos. 43-
3F9, Microfabrication Deposit No. FERM P-13334,4
2-5D11, Microfabrication Deposit No. FERM P-1314
6) was dialyzed against a 0.1 M phosphate buffer (pH 6.5), and S-acetylmercaptosuccinic anhydride at 100 times the molar amount of IgG contained in the solution was added as a dimethylformamide solution. Incubated for 30 minutes. Next, 0.1 M Tris-HCl buffer (pH
7.0) 100 μl, 0.1 M EDTA solution (pH
6.0) 10 μl, 1 M hydroxylamine solution (pH
7.0) Add 100 μl, leave at 30 ° C. for 5 minutes, and
0.1 M phosphate buffer (pH 6.
Gel filtration was carried out using Sephadex G-25 equilibrated in 0) to obtain SH group-labeled mouse anti-human pro-MMP-2 IgG.

2) Preparation of maleimide-labeled peroxidase (POD) POD was dissolved in a 0.1 M phosphate buffer (pH 7.0) so as to have a concentration of 10 mg / ml, and the POD amount was 25 times. A molar amount of N- (ε-maleimidocaproyloxy) succinimide (EMCS) was added as a dimethylformamide solution and reacted at 30 ° C. for 30 minutes.
This reaction solution was subjected to gel filtration using a Sephadex G-25 column equilibrated with a 0.1 M phosphate buffer (pH 6.0) to collect a maleimide-labeled POD fraction.

3) Preparation of IgG-POD complex One mol of the SH group-labeled IgG prepared in the above 1) and about 5 mol of the maleimide-labeled POD obtained in the above 2) were added.
C. and left for 20 hours. This mixture was mixed with U1trogel A equilibrated with 0.1 M phosphate buffer (pH 6.5).
Gel filtration was performed on a cA44 column, and mouse anti-human proMM
The P-2 IgG-POD complex fraction was collected. BSA
And chlorhexidine at 0.1% and 0.00, respectively.
It was added to 1% and stored at 4 ° C.

Example 6 Method for Quantifying Human Pro-MMP-2 (a) Preparation of Monoclonal Antibody-Binding Carrier Immunoassay 4, 209-327 (1
983), according to the method of Ishikawa et al.
Mouse anti-human pro-MMP-2 IgG (clone No.
75-7F7, Deposit No. FERM P-1333
5) were dissolved in 0.1 M phosphate buffer (pH 7.0) containing 0.1% sodium azide, and 100 µg / ml
( _A280 = 0.15). The monoclonal antibody solution was added to a 96-well microplate at 100 μl per well, and left at 4 ° C. for 24 hours. Next, the monoclonal antibody solution was removed and each was diluted with physiological saline for 2 hours.
After washing twice, 1% BSA-0.1M containing 0.1M sodium chloride
It was immersed in 0 mM phosphate buffer (pH 7.0) and stored at 4 ° C.

(B) One-step sandwich EIA method: 1% BSA, 0.1 M sodium chloride and 10 mM
A sample containing purified human pro-MMP-2 or human MMP-2 diluted with 30 mM phosphate buffer (pH 7.0, buffer A) containing EDTA was placed in a 96-well vinyl plate (Fal
con). Next, the enzyme-labeled antibody prepared in Example 5 was diluted with buffer A so as to have a concentration of 1000 ng / ml.
And mixed. 100 μl of this mixture was added to the antibody binding plate prepared in (a) above, reacted at room temperature for 1 hour, and washed four times with physiological saline. Next, a 0.1 M citrate-phosphate buffer containing 0.02% hydrogen peroxide (pH 4.
100 μl of 2 mg / ml o-phenylenediamine dissolved in 9) was added per well, and after reaction at room temperature for 30 minutes, 100 μl of 2N sulfuric acid was added to stop the reaction. A _{492 of} this reaction mixture was transferred to a microplate reader (M
PR-A4 Toyo Soda), and the amount of human MMP-2 in the sample was determined from a calibration curve.

Table 5 shows the above procedures. According to the plate method described in Table 5, clone Nos. 43-3F
A one-step sandwich EIA was performed using the IgG-POD complex from 9,42-5D11 and the antibody-bound carrier prepared in section (a) above (see Table 6 and FIG. 1). In both cases, the absorbance was dependent on the concentration of the standard antigen, but the solid-phase antibody from 75-7F7 did not
gG, IgG-POD complex from 43-3F9 (-.
The-) system has a better solid phase antibody than I-7 from 75-7F7.
IgG, IgG-POD complex from 42-5D11 (-
X-) was observed to show higher absorbance than the system. At this time, clone No. was added to the former IgG-POD complex. 4
In the system using the antibody from 3-3F9, the standard antigen 1-5
A straight line was observed up to a concentration of 00 ng / ml (10-5,000 pg / well). As a result, the clone No.
s. Antibodies from 75-7F7 and 43-3F9 were to be used in the following immunoassays. Clone N
o. Since the antibody from 43-3F9 is a monoclonal antibody against the peptide at the N-terminal side of the human MMP-2 polypeptide, it can be referred to as a pro-MMP-2 measurement system here.

(C) Selection of Buffer Solution The buffer solution used in the one-step sandwich EIA method was examined. Buffer A, 1% BSA,
0.15 M sodium chloride and 10 mM EDTA
A 50 mM Tris-HCl buffer (pH 7.0, buffer B) was used.

According to the method described in Example 6, (b),
20 μl of the standard antigen diluted with buffer A and buffer B was added to a 96-well vinyl plate. Next, the enzyme-labeled antibody prepared in Example 5, IgG (clone No. 43-3F)
9) The -POD complex was diluted with buffer A and buffer B so as to have a concentration of 1000 ng / ml, added to the vinyl plate in an amount of 100 µl each, and mixed. 100 μl of this mixture was added to the antibody-binding plate prepared in Example 6, (a), and reacted at room temperature for 1 hour. The following operations were performed in Example 6,
The operation was performed in the same manner as described in (b). The results of the calibration curve are shown in Table 7 and FIG. Buffer A (-.-), B (-x
-) In each case, the absorbance was dependent on the concentration of the standard antigen, but the buffer A showed a higher absorbance.
As a result, Buffer A was used below.

(D) Dilution test A dilution test was performed according to the method described in Examples 6 and (b). Standard antigen diluted in buffer A or 1/1 to 1
/ 3 kinds of specimens diluted 256 times (normal serum)
Was added to a 96-well vinyl plate in an amount of 10 μl. The following operations were performed in the same manner as described in Example 6, (b), and the diluted serum was measured for each (see Table 8A and FIG. 3). All sera used for the dilution test showed a sufficient straight line, and the regression line passed almost zero points (see Table 8B and FIG. 3).

(E) Simultaneous Reproducibility Test and Sensitivity According to the method described in Example 6 and (b), a simultaneous reproducibility test was performed on a standard antigen solution and a sample (normal human serum) (see Table 9). It was found that the CV value of both the absorbance of the standard antigen solution and the measured serum value was 10% or less.

The average (M) and the standard deviation (SD) when the absorbance of 0 ng / ml of the standard antigen was measured eight times were calculated.
When a standard antigen concentration corresponding to + 2SD is used as the sensitivity, the sensitivity is about 0.24 ng / ml (2.4 pg / well).
l).

(F) Addition and recovery test A standard antigen solution (0, 63, 250,
(500, 1000, 2000 ng / ml), and 10 μl each of them was used as a sample, and 100 μl of an enzyme-labeled antibody solution (1100 ng / ml) was added. Example 6 except that the concentration of o-phenylenediamine was 0.2 mg / ml.
The amount of proMMP-2 was measured in the same manner as in the procedure described in (b), and the amount of the recovered standard antigen was calculated (see Table 10). The average recovery of the standard antigen amount was 99.5%, indicating that a sufficient recovery ratio was obtained and that the antigen amount in 10 μl could be read as an accurate value.

(G) Measurement of Serum Human Pro-MMP-2 Content by One-Step Sandwich EIA Method Serum pro-MMP-2 of various diseases was measured as a sample by the one-step sandwich EIA method described in (b) above (Table 1). 11 and FIG. 4). Healthy human serum (n = 2
13) The pro-MMP-2 value was 570 ng / ml ± 11
It was 8 ng / ml (M ± SD). Serum pro-MMP in patients with thyroid dysfunction, primary liver cancer and biliary cirrhosis
-2 concentrations of 749 ± 166 ng / ml and 686 ± 2, respectively.
The values were 36 ng / ml and 716 ± 135 ng / ml, which were significantly higher than the serum proMMP-2 concentration in healthy subjects. On the other hand, RA, osteoarthritis, gastric cancer and pancreatic cancer patients serum pro-MMP-2 concentration was 408 ± 139 n
g / ml, 449 ± 72 ng / ml, 427 ± 103 n
g / ml and 422 ± 130 ng / ml, which were significantly lower than the serum proMMP-2 concentration in healthy subjects. The serum pro-MMP-2 concentration in patients with chronic pancreatitis had no significant difference compared to that of healthy subjects.

Example 7 Analysis of Serum Antigen An antigen in a sample (serum) was analyzed by immunoblotting. (A) Preparation of Material The sample (normal human serum) was applied to an anti-human pro-MMP-2 monoclonal antibody (clone No. 75-7F7) -bound column, washed sufficiently with Tris-Ca Buffer,
The adsorbed protein was converted to Tris-Ca Bu containing 8M urea.
eluted with effer. The eluted fraction was exchanged for Tris-Ca Buffer using a PD-10 column, and then concentrated using Centricon 10 (Amicon) to obtain a sample for immunoblotting.

(B) Immunoblotting The serum sample prepared in the above (a) was subjected to SDS-PAGE, followed by cell engineering 1 & 2, 1061-1068 (19).
83) Western blotting was carried out according to the method of Tabe described in (8). Next, anti-human MMP-2 IgG (claw) Nos. 43-3F9 and 75-7F7) -P
OD complex, anti-human TIMP-2 1gG (clone N
o. 67-4H11, Deposit No. FERM P-1
2690) -POD complex was used for immunostaining. The anti-human TIMP-2 monoclonal antibody (clone No. 67-4H11) used here was a human TIM
P-2 polypeptide (YRGAAPPKQEFLDIE
An antibody against D) was prepared according to the method described in Example 2, except that KLH was used as the carrier protein for the immunogen. Among the monoclonal antibodies thus obtained,
Clone No. The antibody of 67-4H11 was used as an enzyme-labeled antibody and the antibody-conjugated Seph described in Example 8, (a) below.
Used for arose 4B column.

FIG. 5 shows the results. 43-3F9 and 7
When stained with the 5-7F7 IgG-POD complex (lane 1 and lane 2 respectively), a positive band near 72 kDa, and when stained with the 67-4H11 IgG-POD complex (lane 3), around 24 kDa A positive band was confirmed. Therefore, in serum, MM
It was suggested that P-2 almost existed as a latent (pro) form, and that pro-MMP-2 existed alone or in a complex with TIMP-2.

Example 8 Effect of Human TIMP-2 Addition A fixed amount of pro-MMP-2 to which various concentrations of TIMP-2 were added was used as a sample, and a one-step sandwich EIA was used.
The effect on the system was investigated.

(A) Purification of human placenta-derived human TIMP-2 The placenta was minced and 1 mM calcium chloride, 0.1 M sodium chloride, 0.005% bridge-35, 1 mM N-
Ethylmaleimide, 20 mM Tris-HCl buffer containing 5 mM EDTA, 5 mM benzamidine hydrochloride (pH 7.
4) was added and stirred, and the mixture was allowed to stand for 30 minutes. Then, 10,000 rpm by high-speed cooling centrifuge (HITACHI)
and centrifuged at 4 ° C. for 50 minutes to obtain a supernatant. The same operation was repeated once, and the supernatant was pooled. next,
This supernatant was separated with an anti-human TIMP-2 monoclonal antibody (clone No. 67-4H11) -conjugated Sepharose.
After adsorbing to a 4B column, 1 mM calcium chloride, 0.1 M sodium chloride, 0.005% bridge-
35-containing 20 mM Tris-HCl buffer (pH 7.4, buffer C), its antibody-bound Sepharose 4
The B resin was washed on a glass filter. Furthermore, buffer C and 1 mM calcium chloride, 0.1 M sodium chloride,
After alternately washing with 0.1 M acetate buffer (pH 5.5) containing 0.005% bridge-35, the resin was repacked into the column, and the adsorbed protein was washed with 1 mM calcium chloride, 0.005%.
% Bridge-35 containing 0.1 M glycine-HCl buffer
(PH 2.5) and immediately neutralized with a 3M Tris-HCl buffer (pH 7.5) containing 1 mM calcium chloride and 0.005% bridge-35. Next, this eluted fraction was concentrated by ultrafiltration (Toyo filter paper UHP-43),
Ultragel AcA54 (IBF Biotec)
hnics). A of each fraction
₂₈₀ was measured and the second peak was collected. SDS-P
On AGE, the resulting human TIMP-2 was purified into a single band.

(B) Effect of adding human TIMP-2 The amount of human TIMP-2 was fixed, and the molar ratio of human TIMP-2 (TIMP-2 / proMMP-2) was 125, 25,
1, 0.2, 0.04, and 0 were added. After the reaction, the amount of pro-MMP-2 was diluted with buffer A from which EDTA was removed based on the amount of pro-MMP-2, and the amount of pro-MMP-2 was measured by the one-step sandwich EIA method described in Example 6, (b) (see Table 12). ). As a result, even when the amount of human TIMP-2 added was increased, no change was observed in the _A492 value of each concentration of pro-MMP-2. Therefore, this assay system allows the proMMM in the proMMP-2-TIMP-2 complex
It turned out that P-2 can also be recognized with the same immunoreactivity as free pro-MMP-2.

[0063]

[Table 1]

[0064]

[Table 2]

[0065]

[Table 3]

[0066]

[Table 4]

[0067]

[Table 5]

[0068]

[Table 6]

[0069]

[Table 7]

[0070]

[Table 8]

[0071]

[Table 9]

[0072]

[Table 10]

[0073]

[Table 11]

[0074]

[Table 12]

[Brief description of the drawings]

FIG. 1 is a graph showing a standard curve of human pro-MMP-2, in which the vertical axis represents absorbance at 492 nm and the horizontal axis represents pro-MMP.
The MP-2 concentration (ng / ml) is shown.

FIG. 2. One-step sandwich EI of pro-MMP-2
2 is a graph showing the effect of a buffer used in Method A, in which both the vertical and horizontal axes are the same as in FIG.

FIG. 3 is a graph showing the results of a dilution test of healthy human serum, in which the vertical axis indicates the amount of pro-MMP-2 (ng / ml), and the horizontal axis indicates the dilution ratio (fold).

FIG. 4 is a graph showing the results of measurement of pro-MMP-2 in serum from various diseases, in which the vertical axis indicates the amount of pro-MMP-2 (ng / m
l), the horizontal axis represents each disease number shown in Table 11.

FIG. 5 is a diagram showing the results of immunoblotting of serum antigens.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Hideaki Tokai 11-1, Yokota-Honcho, Takaoka-shi, Toyama Prefecture (72) Inventor Taro Hayakawa 3-406, Mukogaoka, Tenpaku-ku, Nagoya-shi, Aichi Prefecture (72) Inventor Kazushi Iwata 190, Igari-Higashi-cho, Takaoka City, Toyama Prefecture (56) References JP-A-2-1553 (JP, A) JP-A-60-2187 (JP, A) JP-A-4-183397 (JP, A) JP-A-2 -1553 (JP, A) Tokuyo 4-501423 (JP, A) Tokuyo 4-505696 (JP, A) Experimental Medicine Vol. 7, No. 5, P32-40 (1989) (58) Int.Cl. ⁷ , DB name) G01N 33/53 G01N 33/573 G01N 33/577

Claims (2)

(57) [Claims] 1. Two types of monoclonal antibodies that specifically bind to human 72-kDa gelatinase / IV type collagenase, either as an antibody to be bound to a solid-phase carrier or an antibody to be labeled. N of human 72-kDa gelatinase / type IV collagenase
Human 72-k, wherein immunoassay is carried out by a sandwich method using an antibody that recognizes a terminal region.
A method for quantifying Da gelatinase / type IV collagenase. 2. The method for quantifying human 72-kDa gelatinase / IV collagenase according to claim 1, wherein human 72-kDa gelatinase / IV collagenase is measured in serum.